Determining the identity and total number of viable organisms in a particular sample is of tremendous importance. Of specific importance is monitoring and ensuring the safety of food and water supplies through the surveillance and identification of pathogenic organisms in foods and in the environment quickly, efficiently, and accurately.
One such method to accomplish this is the total viable organism (TVO) assay. The TVO assay is widely used today as a quality control application in the industrial microbiology field. The TVO assay is used, for example, to monitor the number and types of bacteria in consumer food products, such as meat. The TVO method can also be used to monitor bacterial populations in drinking water. Monitoring for food and water is, of course, critical to ensure that the food and water supply is safe for consumption.
The steps of the TVO assay generally include: 1) obtaining a test sample; and 2) culturing or plating the sample on agar (a gelatinous nutrient substance), placed in a suitable container. The microbial organisms are allowed to grow and the colony forming units (CFUs) are calculated based on the number of colonies that form on the agar. CFUs can be calculated only after allowing time for colony growth. Samples are typically diluted and this dilution factor (i.e., volume ratio of sample to total volume) is taken into account when calculating CFUs.
Samples can also be cultured on a variety of agar plates that contain different types of selective media to help isolate target microorganisms and more accurately and reliably determine what types of microorganisms are present. Selective agents (e.g., antibiotics, anti-fungals, etc.) will eliminate certain non-target microorganisms (e.g., bacteria of no interest). This avoids the possibility of spurious results that might occur if colonies from many different types of microorganisms are formed.
Selective agents can also favor the growth of certain types of microorganisms over others. Although the TVO assay allows for detection of different microorganism species, e.g., different bacterial species, food and environmental microbiologists must often choose between enumeration and identification without the option of both. Although selective agents can be added to favor the growth of a specific group of organisms, the TVO assay is often based on the ability of normal healthy cells to multiply in nutrient-rich medium (i.e., without selection). TVO therefore has the capacity to measure the total number of microorganisms or a group of microorganisms in the sample tested. However, because of the lack of ability to differentiate specific microorganisms, TVO can be relatively nonspecific for the microorganism population as a whole.
There are numerous other methods available that identify specific microorganisms, especially pathogens. Such methods are widely used in the clinical setting. Methods for detecting microorganisms often depend upon enrichment of the microorganism culture in order to increase the numbers of the target microorganism and to allow for the resuscitation of injured microorganisms. When selective and differential plating is employed, researchers are able to discriminate the target organism from the background microflora. However, the results are almost always non-enumerative. In other words, only the presence or absence of a particular bacterial population can be determined, not the quantity.
Utilizing both sample enrichment and selective plating results is a time-consuming assay, which often takes several days before even a preliminary result can be obtained. Although such enrichment and selective plating is a staple procedure to determine the number and types of microorganisms in a sample, it can typically take several days to get a final result after colonies grown on agar are counted. The amount of time it takes to obtain results is the most significant drawback of using the staple TVO assay.
Different methods have been developed that attempt to shorten detection time by eliminating the selective and differential plating steps. Such methods include DNA hybridization, agglutination, and enzyme immunoassay. Although these alternative techniques have shortened the time for detection, culture enrichment steps remain necessary because these methods only allow for the ultimate detection of 103-104 CFU of the target pathogen. Therefore, confirmation for presumptively positive results remains necessary for the TVO assay.
Furthermore, there is no universal method or single technique available for analyzing a biological sample, especially a food sample, to detect for the presence or absence of multiple microorganisms. This makes the sample preparation steps for the separation and subsequent concentration of microorganisms from a biological sample prior to assay for the microorganisms a rate limiting step in molecular methods for the detection of pathogens, including foodborne pathogens.
With regard to specific sample preparation techniques for separation of microorganisms, techniques that utilize centrifugation followed by washing and filtration steps are not advantageous because they result in a significant loss of, or damage to, microorganisms during the processing. Furthermore, the whole procedure is not amenable for automation.
In order to achieve separation of the microorganism from the sample, affinity agents for a particular microorganism have been employed. However, affinity agents used to isolate microorganisms from the complex matrices are also complicated to deploy because of: 1) lack of universal affinity agents that bind to all organisms selectively from the other sample constituents; 2) variability in binding affinities of different organisms to the universal affinity reagents; and 3) difficulty in eluting the bound organism back into the solution.
Other techniques for identifying pathogens in food and water are also known. For example, flow cytometry has been reported as a rapid technique for enumerating and identifying microorganisms. Flow cytometry is a method originally used to separate and analyze eukaryotic cell populations but has been employed in the evaluation and detection of microorganisms, as well. Specifically, microorganisms that have been fluorescently stained, for example with a nucleic acid dye, are passed through a beam of light. A pattern unique to the microorganism of interest is achieved by the combination of both the adsorption and scattering of the light. (Breeuwer et al., Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in S. cerevisiae, which result in accumulation of fluorescent product, Appl. Environ. Micriobiol., 61(4):1614-9 (April 1995); de Boer & Beumer, Methodology for detection and typing of foodborne microorganisms, Int. J. Food. Microbiol., 50(1-2):119-30 (September 1999)).
The main advantage of flow cytometry is that it is fast and easy to perform. Flow cytometry is adaptable to different types of samples and methods, making it a robust application that is also amenable to automation. It is no surprise that numerous flow cytometry applications have emerged in industrial biotechnology, food and pharmaceutical quality control, routine monitoring of drinking water and wastewater systems, and microbial ecological research in soils and natural aquatic habitats. Flow cytometry results correlate well with the results of standard plate counting methods.
However, flow cytometry has other limitations, such as the need to dye label target microorganisms for detection, the high cost of the equipment and the need for specialized training of personnel. Further limits on detection are imposed by interference of nonspecific fluorescence, less than optimal detection limits, difficulty in applying the method to solid or particulate food samples, the inability to differentiate between viable and dead cells unless specialized staining is used, and destruction of cellular viability that may also occur during sample processing (Quintero-Betancourt et al., Cryptosporidium parvum and Cyclospora cayetanensis: a review of laboratory methods for detection of the waterborne parasites, J. Microbiol. Methods, 49(3):209-24 (May 2002)). The extensive and routine use of this technique has begun to alleviate these drawbacks.
Other practical problems remain with flow cytometry, especially in the context of analyzing biological or environmental samples derived from what are referred to as “complex matrices.” Complex matrices may consist of substances, including particulate matter, which interferes with the detection of microorganisms in the biological or environmental sample. The nucleic acid dyes, used to detect the microorganism in the sample, may non-specifically bind to such particulate matter in the sample matrix resulting in a high background fluorescent signal. This high background makes it difficult to identify and analyze low concentrations of microorganism in the sample.
As described in U.S. Pat. No. 7,205,100 to Buttry et al., the entire contents of which are hereby incorporated by reference herein, fluorescent background and fluorescent signals generated by membrane permeable dead cells can be reduced by mixing target specific fluorescent dyes and fluorescence quenchers in the samples of interest. However, in some sample matrices containing extracellular particles that have non-specific affinity to the target specific fluorescent dyes, fluorescent background cannot be quenched effectively by fluorescent quenchers. In addition, in samples containing large amount of extracellular particles that have non-specific affinity to the target specific fluorescent dyes, the vast majority of target specific dyes will bind to the particles non-specifically, and as a result not enough dye molecules are available in the solution to label the target organisms.
Consequently, methods that address the drawbacks in current methods for detecting the presence or absence of microorganisms in a sample using flow cytometry are sought.